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Plant Proteomics Methods and Protocols Methods in Molecular Biology 2139 3rd Edition Jesus V. JorrinNovo
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Preface
Proteomics techniques have constantly been developed further through the last decade and have been applied successfully for all kinds of samples and biological or medical questions. Nowadays, they are established methods in many research labs, and proteomic studies can be accomplished with good reliability and coverage on a routine basis. Therefore, proteomics can be used as a powerful tool in functional genomics and systems biology studies. Current challenges are thus the implementation of proteomic analyses in these comprehensive studies. This applies for both sample generation and preparation to ensure consistency over several levels of analyses like genomics, transcriptomics, and metabolomics and integration of the multilevel data to generate biological knowledge. This book gives an overview of contemporary quantitative proteomics methods and data interpretation approaches and also gives examples of how to implement proteomics into systems biology.
Germany Jörg Reinders
Regensburg,
Preface .
Contributors.
1 Multiplexed Quantitative Proteomics for High-Throughput Comprehensive Proteome Comparisons of Human Cell Lines. .
Amanda Edwards and Wilhelm Haas
2 Sample Preparation Approaches for iTRAQ Labeling and Quantitative Proteomic Analyses in Systems Biology.
Christos Spanos and J. Bernadette Moore
3 Two Birds with One Stone: Parallel Quantification of Proteome and Phosphoproteome Using iTRAQ .
Fiorella A. Solari, Laxmikanth Kollipara, Albert Sickmann, and René P. Zahedi
4 Selected Reaction Monitoring to Measure Proteins of Interest in Complex Samples: A Practical Guide.
Yuehan Feng and Paola Picotti
5 Monitoring PPARG-Induced Changes in Glycolysis by Selected Reaction Monitoring Mass Spectrometry.
Andreas Hentschel and Robert Ahrends
6 A Targeted MRM Approach for Tempo-Spatial Proteomics Analyses.
Annie Moradian, Tanya R. Porras-Yakushi, Michael J. Sweredoski, and Sonja Hess
7 Targeted Phosphoproteome Analysis Using Selected/Multiple Reaction Monitoring (SRM/MRM)
Jun Adachi, Ryohei Narumi, and Takeshi Tomonaga
8 Testing Suitability of Cell Cultures for SILAC-Experiments Using SWATH-Mass Spectrometry.
Yvonne Reinders, Daniel Völler, Anja-K. Bosserhoff, Peter J. Oefner, and Jörg Reinders
9 Combining Amine-Reactive Cross-Linkers and Photo-Reactive Amino Acids for 3D-Structure Analysis of Proteins and Protein Complexes
Philip Lössl and Andrea Sinz
10 Tissue MALDI Mass Spectrometry Imaging (MALDI MSI) of Peptides
Birte Beine, Hanna C. Diehl, Helmut E. Meyer, and Corinna Henkel
11 Ethyl Esterification for MALDI-MS Analysis of Protein Glycosylation.
Karli R. Reiding, Emanuela Lonardi, Agnes L. Hipgrave Ederveen, and Manfred Wuhrer
12 Characterization of Protein N-Glycosylation by Analysis of ZIC-HILIC-Enriched Intact Proteolytic Glycopeptides.
Gottfried Pohlentz, Kristina Marx, and Michael Mormann
13 Simple and Effective Affinity Purification Procedures for Mass Spectrometry-Based Identification of Protein-Protein Interactions in Cell Signaling Pathways. . .
Julian H.M. Kwan and Andrew Emili
14 A Systems Approach to Understand Antigen Presentation and the Immune Response
Nadine L. Dudek, Nathan P. Croft, Ralf B. Schittenhelm, Sri H. Ramarathinam, and Anthony W. Purcell
15 Profiling of Small Molecules by Chemical Proteomics .
Kilian V.M. Huber and Giulio Superti-Furga
16 Generating Sample-Specific Databases for Mass Spectrometry-Based Proteomic Analysis by Using RNA Sequencing
Toni Luge and Sascha Sauer
17 A Proteomic Workflow Using High-Throughput De Novo Sequencing Towards Complementation of Genome Information for Improved Comparative Crop Science
Reinhard Turetschek, David Lyon, Getinet Desalegn, Hans-Peter Kaul, and Stefanie Wienkoop
18 From Phosphoproteome to Modeling of Plant Signaling Pathways .
Maksim Zakhartsev, Heidi Pertl-Obermeyer, and Waltraud X. Schulze
19 Interpretation of Quantitative Shotgun Proteomic Data
Elise Aasebø, Frode S. Berven, Frode Selheim, Harald Barsnes, and Marc Vaudel
20 A Simple Workflow for Large Scale Shotgun Glycoproteomics.
Astrid Guldbrandsen, Harald Barsnes, Ann Cathrine Kroksveen, Frode S. Berven, and Marc Vaudel
21 Systemic Analysis of Regulated Functional Networks.
Luis Francisco Hernández Sánchez, Elise Aasebø, Frode Selheim, Frode S. Berven, Helge Ræde, Harald Barsnes, and Marc Vaudel Index
Contributors
ELISE AASEBØ • Proteomics Unit, Department of Biomedicine, University of Bergen, Bergen, Norway
JUN ADACHI • Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan
ROBERT AHRENDS • Leibniz-Institut für Analytische Wissenschaften—ISAS—e.V., Dortmund, Germany
HARALD BARSNES • Proteomics Unit, Department of Biomedicine, University of Bergen, Bergen, Norway; KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway
BIRTE BEINE • Leibniz-Institut für Analytische Wissenschaften—ISAS—e.V., Dortmund, Germany
FRODE S. BERVEN • Proteomics Unit, Department of Biomedicine, University of Bergen, Bergen, Norway; KG Jebsen Centre for Multiple Sclerosis Research, Department of Clinical Medicine, University of Bergen, Bergen, Norway; Norwegian Multiple Sclerosis Competence Centre, Department of Neurology, Haukeland University Hospital, Bergen, Norway
ANJA-K. BOSSERHOFF • Institute of Pathology, University of Regensburg, Regensburg, Germany; Institute of Biochemistry, Emil-Fischer-Zentrum, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany
NATHAN P. CROFT • Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC, Australia
GETINET DESALEGN • Department of Crop Sciences, University of Natural Resources and Life Sciences, Vienna, Austria
HANNA C. DIEHL • Clinical Proteomics, Medizinisches Proteome-Center, Ruhr-University, Bochum, Germany
NADINE L. DUDEK • Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC, Australia
AMANDA EDWARDS • Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA; Department of Medicine, Harvard Medical School, Boston, MA, USA
ANDREW EMILI • Department of Molecular Genetics, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada
YUEHAN FENG • Department of Biology, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland
ASTRID GULDBRANDSEN • Proteomics Unit, Department of Biomedicine, University of Bergen, Bergen, Norway; KG Jebsen Centre for Multiple Sclerosis Research, Department of Clinical Medicine, University of Bergen, Bergen, Norway
WILHELM HAAS • Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA; Department of Medicine, Harvard Medical School, Boston, MA, USA
CORINNA HENKEL • Leibniz-Institut für Analytische Wissenschaften—ISAS—e.V., Dortmund, Germany
ANDREAS HENTSCHEL • Leibniz-Institut für Analytische Wissenschaften—ISAS—e.V., Dortmund, Germany
LUIS FRANCISCO HERNÁNDEZ SÁNCHEZ • Graduate Program in Optimization, Universidad Autónoma Metropolitana Azcapotzalco, Mexico City, Mexico
SONJA HESS • Proteome Exploration Laboratory, California Institute of Technology, Pasadena, CA, USA
AGNES L. HIPGRAVE EDERVEEN • Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands
KILIAN V.M. HUBER • Structural Genomics Consortium, University of Oxford, Oxford, UK; Target Discovery Institute, University of Oxford, Oxford, UK
HANS-PETER KAUL • Department of Crop Sciences, University of Natural Resources and Life Sciences, Vienna, Austria
LAXMIKANTH KOLLIPARA • Leibniz-Institut für Analytische Wissenschaften—ISAS—e.V., Dortmund, Germany
ANN CATHRINE KROKSVEEN • Proteomics Unit, Department of Biomedicine, University of Bergen, Bergen, Norway; KG Jebsen Centre for Multiple Sclerosis Research, Department of Clinical Medicine, University of Bergen, Bergen, Norway
JULIAN H.M. KWAN • Department of Molecular Genetics, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada
EMANUELA LONARDI • Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands
PHILIP LÖSSL • Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Halle/Saale, Germany; Biomolecular Mass Spectrometry and Proteomics, Netherlands Proteomics Center, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
TONI LUGE • Otto Warburg Laboratory, Max Planck Institute for Molecular Genetics, Berlin, Germany
DAVID LYON • Department of Ecogenomics and Systems Biology, University of Vienna, Vienna, Austria
KRISTINA MARX • Bruker Daltonik GmbH, Bremen, Germany
HELMUT E. MEYER • Leibniz-Institut für Analytische Wissenschaften—ISAS—e.V., Dortmund, Germany
J. BERNADETTE MOORE • Department of Nutritional Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford Surrey, UK
ANNIE MORADIAN • Proteome Exploration Laboratory, California Institute of Technology, Pasadena, CA, USA
MICHAEL MORMANN • Institute for Hygiene, University of Münster, Münster, Germany
RYOHEI NARUMI • Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, Kobe, Japan
PETER J. OEFNER • Institute of Functional Genomics, University of Regensburg, Regensburg, Germany
HEIDI PERTL-OBERMEYER • Plant Systems Biology, Plant Physiology, University of Hohenheim, Stuttgart, Germany
PAOLA PICOTTI • Department of Biology, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland
GOTTFRIED POHLENTZ • Institute for Hygiene, University of Münster, Münster, Germany
Contributors
TANYA R. PORRAS-YAKUSHI • Proteome Exploration Laboratory, California Institute of Technology, Pasadena, CA, USA
ANTHONY W. PURCELL • Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC, Australia; The Department of Biochemistry and Molecular Biology, The Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, VIC, Australia
HELGE RÆDER • KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway; Department of Pediatrics, Haukeland University Hospital, Bergen, Norway
SRI H. RAMARATHINAM • Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC, Australia
KARLI R. REIDING • Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands
JÖRG REINDERS • Institute of Functional Genomics, University of Regensburg, Regensburg, Germany
YVONNE REINDERS • Department of Biochemistry I, University of Regensburg, Regensburg, Germany
SASCHA SAUER • Otto Warburg Laboratory, Max Planck Institute for Molecular Genetics, Berlin, Germany
RALF B. SCHITTENHELM • Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC, Australia
WALTRAUD X. SCHULZE • Plant Systems Biology, Plant Physiology, University of Hohenheim, Stuttgart, Germany
FRODE SELHEIM • Proteomics Unit, Department of Biomedicine, University of Bergen, Bergen, Norway
ALBERT SICKMANN • Leibniz-Institut für Analytische Wissenschaften—ISAS—e.V., Dortmund, Germany; Department of Chemistry, College of Physical Sciences, University of Aberdeen, Aberdeen, Scotland, UK
ANDREA SINZ • Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Halle/Saale, Germany
FIORELLA A. SOLARI • Leibniz-Institut für Analytische Wissenschaften—ISAS—e.V., Dortmund, Germany
CHRISTOS SPANOS • Department of Nutritional Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford Surrey, UK
GIULIO SUPERTI-FURGA • CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
MICHAEL J. SWEREDOSKI • Proteome Exploration Laboratory, California Institute of Technology, Pasadena, CA, USA
TAKESHI TOMONAGA • Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan
REINHARD TURETSCHEK • Department of Ecogenomics and Systems Biology, University of Vienna, Vienna, Austria
MARC VAUDEL • Proteomics Unit, Department of Biomedicine, University of Bergen, Bergen, Norway
DANIEL VÖLLER • Institute of Pathology, University of Regensburg, Regensburg, Germany
STEFANIE WIENKOOP • Department of Ecogenomics and Systems Biology, University of Vienne, Vienna, Austria
MANFRED WUHRER • Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands; Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, The Netherlands
RENÉ P. ZAHEDI • Leibniz-Institut für Analytische Wissenschaften—ISAS—e.V., Dortmund, Germany
MAKSIM ZAKHARTSEV • Plant Systems Biology, Plant Physiology, University of Hohenheim, Stuttgart, Germany
Chapter 1
Multiplexed Quantitative Proteomics for High-Throughput Comprehensive Proteome Comparisons of Human Cell Lines
Amanda Edwards and Wilhelm Haas
Abstract
The proteome is the functional entity of the cell, and perturbations of a cellular system almost always cause changes in the proteome. These changes are a molecular fingerprint, allowing characterization and a greater understanding of the effect of the perturbation on the cell as a whole. Monitoring these changes has therefore given great insight into cellular responses to stress and disease states, and analytical platforms to comprehensively analyze the proteome are thus extremely important tools in biological research. Mass spectrometry has evolved as the most relevant technology to characterize proteomes in a comprehensive way. However, due to a lack of throughput capacity of mass spectrometry-based proteomics, researchers frequently use measurement of mRNA levels to approximate proteome changes. Growing evidence of substantial differences between mRNA and protein levels as well as recent improvements in mass spectrometry-based proteomics are heralding an increased use of mass spectrometry for comprehensive proteome mapping. Here we describe the use of multiplexed quantitative proteomics using isobaric labeling with tandem mass tags (TMT) for the simultaneous quantitative analysis of five cancer cell proteomes in biological duplicates in one mass spectrometry experiment.
Key words Quantitative proteomics, Multiplexing, Isobaric labels, TMT
1 Introduction
Proteins are the primary functional units of the cell, and as such, information about their abundance, interaction partners, and modifications is critical for understanding both healthy and abnormal cellular function. Traditionally, such work has been accomplished on a protein-by-protein basis through genetic or biochemical techniques. More recently, large-scale approaches attempting to monitor an entire proteome—all proteins expressed in a cell or tissue—in one step have become accessible [1, 2]. Such a holistic approach allows identification of proteome imbalances and changes in functional networks, enabling us to study and probe the state of a cell in an unbiased and rapid fashion.
Mass spectrometry has emerged as the leading platform to rapidly characterize whole proteomes, primarily driven by improvements in both MS sensitivity and throughput in the last 15 years. However, the technology has traditionally lagged behind in throughput capacity when compared to genomics platforms, such as DNA-microarrays or next-generation sequencing technology, to study cellular expression profiles. Therefore, mRNA expression profiles are still the main source for estimations of protein-level changes for most researchers [3, 4]. Yet evidence is accumulating that significant differences exist between mRNA- and protein-level changes in different cell or tissue states [1, 5–9]. There is thus an enormous need for improved mass spectrometry-based proteomics technology to enable direct protein-level measurements, with a throughput comparable to that provided by genomics technologies.
Mass spectrometry-based proteomics has been used as a quantitative tool since the late 1990s with the introduction of accurate relative quantification using stable isotopes. One of the earliest approaches was the employment of isotope-coded affinity tags (ICAT). ICAT enables a chemical incorporation of differential stable isotopes into two different samples and, in parallel, a reduction of proteome complexity by enrichment of cysteine-containing peptides [10]. An approach to incorporate stable isotopes metabolically through heavy isotope-labeled amino acids—stable isotope labeling in cell culture (SILAC)—was first described in 2002 [11] and is still widely used. The commercial availability of highperformance mass spectrometers [12, 13] optimized for use in combination with liquid chromatography further contributed to the propagation of quantitative proteomics, and new strategies for chemical incorporation of stable isotopes into peptides, such as reductive dimethylation [14, 15], arose. Each of the above methods relies on the use of full MS data from intact peptide ions to perform quantitative analysis. Each peptide in the two samples of interest is detected in its light and heavy form, leading to an increase in the signal complexity in the full MS spectra. This increase in signal complexity necessarily decreases the overall sensitivity of the approach and complicates the quantitative analysis of individual peptides. Consequently, although more is theoretically possible [16], the number of samples routinely compared simultaneously using these methods is limited to three [17].
A very elegant strategy to remove this roadblock in multiplexing MS proteomics was first described in 2003 through the use of isobaric tags to incorporate stable isotopes into proteomics samples [18]. These tags consist of three regions: a mass reporter ion, a mass balancer region, and a reactive terminal amino group. To quantify different protein levels in different biological samples, peptide mixtures are labeled with different forms of the tag by allowing the tag to react with amino groups at the N-terminus or lysine
residues of a peptide. Importantly, each tag has the same mass, as the chemical structures only differ in the distribution of heavy stable isotopes between reporter and balancer regions. Thus differentially labeled peptides migrate together through the chromatographic separation and are indistinguishable in MS1 scans. However, during MS2 fragmentation, the mass reporter ions (with a unique mass for each tag) separate from the parent tag, and their relative intensities represent the relative abundance of the original peptides in the measured samples. There are two commercial sources for isobaric tags: isobaric tags for relative and absolute quantitation (ITRAQ) reagents (Sciex) that allow the analysis of up to eight samples [19, 20], and tandem mass tag (TMT) reagents (Pierce) that enable a simultaneous quantification of up to ten samples [21, 22]. An early caveat of the isobaric labeling strategy was a limitation in the achievable accuracy and reproducibility of quantitative results due to coisolation and fragmentation of contaminant ions with the ions targeted for identification and quantification. Solutions to overcome this limitation were presented in the form of applying ion-ion chemistry for removing contaminant ions [23] or by separating identification and quantification of a peptide ion, performing the identification based on MS2 data but shifting the quantification to an MS3 experiment as an additional gas-phase enrichment and fragmentation step [24]. The MS3 method was further optimized to increase sensitivity and throughput, and this MultiNotch MS3 method [25] is now implemented as a synchronous precursor selection (SPS)-supported MS3 method on the Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). We believe that multiplexed quantitative proteomics is a tool that will prove to be indispensable in studying complex biological systems and disease states requiring the analysis of many samples.
This chapter describes the workflow for using 10-plex tandem mass tag (TMT) reagents for isobaric labeling-based multiplexed quantitative proteomics to comprehensively map proteomes of human cell lines. We routinely apply this protocol to quantify approximately 8000 proteins simultaneously in ten samples, occupying 36 h of mass spectrometry time, or less than 4 h to quantitatively characterize the proteome of a human cell line.
2 Materials
2.1 Cell Culture
1. Cell lines: This protocol is applicable for the proteomic analysis of any adherent human cell line. Detached cell lines can also be used, with modifications to the cell culture protocols.
2. Cell media: Use culture media appropriate for the chosen cell lines.
3. 1× sterile phosphate-buffered saline (PBS).
4. 0.25 % Trypsin.
Amanda Edwards and Wilhelm Haas
2.2 Cell Lysis
2.3 Sample
Preparation for Mass Spectrometry
1. Lysis buffer: 75 mM NaCl, 50 mM HEPES (pH 8.5), 10 mM sodium pyrophosphate, 10 mM NaF, 10 mM β-glycerophosphate, 10 mM sodium orthovanadate, 10 mM phenylmethanesulfonylfluoride (PMSF), Roche Complete Protease Inhibitor EDTA-free tablet, 3 % SDS.
1. HPLC-grade methanol.
2. HPLC-grade chloroform.
3. HPLC-grade water.
4. HPLC-grade acetonitrile (ACN).
5. HPLC-grade acetone.
6. 1 M Dithiothreitol (DTT) in 50 mM HEPES (pH 8.5).
7. 1 M Iodoacetamide (IAA) in 50 mM HEPES (pH 8.5).
8. Digestion buffer: 1 M urea, 50 mM HEPES (pH 8.5).
9. Lysyl endopeptidase (LysC) (Wako Chemicals, 10 AU, resuspended in 2 mL HPLC-grade water, stored at −80 °C).
3.3 Reduction, Alkylation, and Precipitation of Proteins
1. Grow each of the five cell lines to 90 % confluence in duplicate in 10 cm2 dishes (a total of ten samples).
2. Prior to collecting the cells, wash gently twice with 2.5 mL pre-warmed sterile 1× PBS (see Note 1).
3. Add 2 mL pre-warmed trypsin to each 10 cm2 dish, covering the cell layer completely. Incubate for 5 min at 37 °C. Add 3 mL pre-warmed media to each 10 cm2 dish, and collect each cell mixture in 15 mL Falcon tubes.
4. Pellet cells by centrifuging at 500 × g for 5 min. Discard the supernatant. Wash the cell pellet once with sterile 1× PBS (see Note 2).
Note: All subsequent steps should be performed at room temperature, as the SDS in the lysis buffer will precipitate at cold temperatures.
1. Resuspend each cell pellet in 0.5 mL lysis buffer, pipetting up and down to disrupt the cell pellet (see Note 3).
2. Lyse the cells by passing the resuspended cells ten times through a 21-gauge needle (see Note 4). Transfer the suspension to 1.5 mL Eppendorf tubes.
3. Clear away cellular debris by centrifuging at 16,000 × g for 5 min. Collect the supernatant (see Note 5).
1. Add 2.5 μL of 1 M DTT to each sample (final concentration of DTT = 5 mM), and vortex thoroughly. Centrifuge briefly at 3000 × g to bring all contents to the bottom of the tube. Incubate at 56 °C for 30 min (see Note 6).
3.1 Cell Culture
Amanda Edwards and Wilhelm Haas
2. Cool the tubes on ice for 3 min.
3. Add 7.5 μL of 1 M IAA to each sample (final concentration of IAA = 15 mM), and vortex thoroughly. Centrifuge briefly at 3000 × g. Incubate in the dark for 20 min (see Note 7).
4. Add 2.5 μL of 1 M DTT to each sample to quench the reaction, and vortex thoroughly. Centrifuge briefly at 3000 × g. Incubate in the dark for 15 min.
5. Transfer each sample to a 15 mL Falcon tube.
6. Begin protein precipitation by adding 2.0 mL methanol to each tube (4 × the initial lysate volume) (see Note 8). Vortex, and centrifuge at 1300 × g for 3 min.
7. Add 0.5 mL chloroform to each tube (1 × the initial lysate volume), and vortex. Make sure to disrupt any pellet fully (see Note 9). Centrifuge at 1300 × g for 3 min.
8. Add 1.5 mL water to each tube (3 × the initial lysate volume), and vortex. Again, make sure that pellet is fully disrupted. Centrifuge at 1300 × g for 3 min.
9. At this stage, precipitated proteins should form a white disk between the aqueous and organic phases. Carefully remove all aqueous and organic supernatant.
10. Wash the protein pellet with 2.0 mL methanol. Centrifuge at 1300 × g for 3 min.
11. Remove the supernatant, and place the protein pellet on ice. Add 1.5 mL ice-cold acetone to each pellet. Disrupt the pellet, and centrifuge at 1300 × g at 4 °C for 3 min. Remove the supernatant, and repeat once (see Note 10).
12. Dry the precipitated protein in open tubes at 56 °C for 15 min or at 37 °C for 60 min, until pellets are completely dry. Cool the pellet on ice for 5 min (see Note 11).
3.4 Digestion of Proteins
1. Resuspend the protein pellet in 0.5 mL digestion buffer (see Note 12).
2. Add 2.5 μL LysC stock (5 μg) to each pellet. Vortex. Centrifuge briefly at 3000 × g. Incubate overnight at room temperature, agitating gently on a tabletop vortexer.
3. Add 12.5 μL trypsin stock (5 μg) to each tube. Vortex. Centrifuge briefly at 3000 × g. Incubate at 37 °C for 6 h.
4. Acidify the reaction with 25 μL 10 % TFA (final concentration TFA = 0.5 %). Vortex. Centrifuge at 16,000 × g for 5 min, and collect the supernatant (see Note 13).
3.5 Cleanup of Sample Using SepPak Columns (See Notes 14 and 15)
1. Place 50 mg C18 SepPak columns on the vacuum manifold, and wash with 5 × 1 mL ACN.
2. Wash with 5 × 1 mL 80 % ACN, 0.5 % acetic acid.
3. Wash with 5 × 1 mL 0.1 % TFA.
3.6 Quantify and Aliquot Digested Peptides
4. Apply sample to column, and pull over column at a slow speed (see Note 16).
5. Wash with 5 × 1 mL 0.1 % TFA.
6. Wash with 1 mL 0.5 % acetic acid, and allow the column to go completely dry.
7. Remove SepPak columns from the vacuum manifold, and place in clean 2 mL Eppendorf tubes. Add 0.75 mL of 40 % ACN, 0.5 % acetic acid to each column. Using a 1 mL syringe, push solution through the column slowly. Add 0.75 mL of 80 % ACN, 0.5 % acetic acid to each column, and push the solution through the column, allowing the column to go completely dry (see Note 17).
8. Dry the eluted peptides in a SpeedVac.
1. Resuspend each sample in 0.5 mL 50 % ACN, 5 % formic acid. Vortex. Centrifuge briefly at 3000 × g. Sonicate for 5 min.
2. (See Note 18) Prepare standard bovine serum albumin (BSA) stocks ranging from 25 to 2000 μg/mL, with 50 % ACN, 5 % formic acid as the buffer.
3. Add 10 μL of BSA standard or sample into 96-well plate wells (see Note 19). Add 200 μL of working reagent to each well, and mix thoroughly.
4. Cover the plate with plastic wrap, and incubate at 37 °C for 30 min. Remove the plate, and allow cooling to room temperature for 5 min.
5. Measure the absorbance at 562 nM, and use the standard curve to determine the protein concentration of each sample.
6. Prepare 50 μg aliquots of each sample, and dry the peptides in a SpeedVac.
3.7 TMT Labeling of Peptides
1. Resuspend the TMT reagent according to the manufacturer’s instructions in anhydrous acetonitrile (see Note 20).
2. Resuspend the peptides in 50 μL 200 mM HEPES (pH 8.5), 30 % anhydrous ACN (see Note 21). Vortex. Centrifuge briefly at 3000 × g. Sonicate for 5 min.
3. Add 5 μL of TMT reagent to each peptide solution, with 1 TMT label used for each of the ten samples (126, 127n, 127c, 128n, 128c, 129n, 129c, 130n, 130c, and 131).
4. Incubate the reaction mixtures at room temperature for 1 h.
5. Quench the reaction by adding 6 μ L of 200 mM HEPES (pH 8.5), 5 % hydroxylamine. Incubate at room temperature for 15 min.
6. Acidify the mixture by adding 50 μL of 1 % TFA. Combine all ten samples into one sample, as they are now all distinctly labeled.
Amanda Edwards and Wilhelm Haas
3.8 Fractionation of Peptides
7. De-salt the mixture over a 50 mg C18 SepPak column (see Subheading 3.5 above for details).
8. Dry the peptides in a SpeedVac.
1. Resuspend the peptides in 0.5 mL 5 % ACN, 5 % formic acid. Vortex. Centrifuge briefly at 3000 × g. Sonicate for 5 min.
2. Fractionate the sample by high pH reverse-phase high-pressure liquid chromatography (HpRP) using a two-buffer gradient system at a flow rate of 500 μL/min. Load the sample in 0 % HpRP buffer B for 2 min, and then separate the peptides using a linear gradient from 20 to 35 % HpRP buffer B over 60 min. Wash the column with 100 % HpRP buffer B for 5 min, and re-equilibrate the column with 100 % HpRP buffer A for 10 min. Monitor peptide elution by UV absorption at a wavelength of 220 nm and collect a total of 96 fractions in a deepwell 96-well plate from 11.5 to 69.5 min (see Note 22).
3. Combine the 96 fractions into 12 fractions, based on the schematic in Fig. 1.
4. Dry the fractions in a SpeedVac.
3.9 Mass Spectrometry Analysis
The details of the LC-MS2/MS3 methods will depend on the instrumentation available. Here, we describe a method using an Easy-nLC 1000 (Thermo Fisher Scientific) with chilled autosampler and an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific)
1. Sample preparation: Resuspend each fraction in 8 μL 5 % ACN, 5 % formic acid and sonicate to ensure full suspension of all peptides. Inject 3 μL of each sample for chromatographic separation and mass spectrometry analysis.
2. Nanospray liquid chromatography method: Separate peptides over a 100 μm inner diameter microcapillary column, packed in-house with 0.5 cm of Magic C4 resin, 0.5 cm of Maccell C18 resin, and 29 cm of GP-C18 resin. Use a 6–25 % gradient of MS buffer B over 165 min at 300 nL/min to elute the peptides. End the gradient with a 10-min wash with 100 % MS buffer B to clear all remaining peptides off the column, and re-equilibrate the column with 9 μL of 100 % MS buffer A to prepare the column for subsequent runs.
3. Mass spectrometry method: Begin acquisition with a full MS1 spectrum acquired in the Orbitrap, and use synchronous precursor selection to isolate the ten highest intensity peptides for MS2 analysis. Following CID fragmentation of these peptides, perform MS2 scans in the linear ion trap. Once again, use synchronous precursor selection to isolate the ten highest intensity peptides for MultiNotch MS3 analysis [25]. Following HCD fragmentation of the peptides, perform MS3 scans in the Orbitrap for maximum sensitivity (see Note 23).
3.10 Data Analysis
As above, the details of the data analysis will depend on the specific search algorithms and software used. While we use Sequest [26] to match peptide spectra to sequences, a variety of other options are available (e.g., Mascot, X!Tandem). However, some parameters should be universally applied.
1. Specific search parameters include digestion enzyme, static peptide modifications, variable peptide modifications, and precursor ion tolerance. In this case, select trypsin as the enzyme,
Fig. 1 An overview of the workflow of a multiplexed quantitative proteomics measurement, from cell culture to mass spectrometer
Amanda Edwards and Wilhelm Haas
requiring all matching peptides to have termini consistent with tryptic cleavage, allowing at most two missed cleavages. Static modifications include the TMT label on the N-terminus and lysine residues (229.162932 Da) as well as carbamidomethylation (57.021464 Da) on cysteine residues. Oxidation of methionine (15.994915 Da) should be set as a variable modification. Set the precursor m/z ion tolerance to 50 ppm.
2. Several online servers provide complete or near-complete protein databases for a variety of species, including UniProt, Ensembl, and RefSeq, against which MS2 spectra can be searched. We use UniProt databases, and we apply a targetdecoy database search strategy to accurately estimate the false discovery rate of peptide and protein identifications [27, 28]. This requires compiling a concatenated database with a target component including the organism-specific protein sequence database as well as that of known contaminants such as porcine trypsin or other proteases used in the sample preparation. The second and so-called decoy component includes the same sequences but in reversed—or flipped—order, where every protein sequence starts with the original C-terminus of the original sequence. A practical protocol for the use of this database for estimating the FDR of a proteomics dataset is described elsewhere [29]. Filter the final results of peptides as well as protein identifications to an FDR of at most 1 %. Several algorithms are used for filtering proteomics data. We use a linear discriminant analysis to assess the FDR of MS2 spectra assignments to peptide sequences (peptide-spectral matches, PSMs). For a protein identification FDR filter, we also rely on the target-decoy database search strategy by using a posterior error histogram with protein FDR estimations that are based on combining probabilities of correct assignments for each PSM for all peptides matching a protein sequence [30].
3. In order to accurately quantify a peptide, signal-to-noise values and isolation specificity must exceed a background threshold. These values will depend on the instrument being used. To quantify a protein level, sum all reporter ion intensities from all peptides assigned to that protein.
4. Normalization of the data allows correction for slight preparation errors or MS anomalies. We recommend a two-step normalization procedure. Begin by normalizing all protein intensities to the ratio of the average intensity of that protein to all median protein intensities. This will bring all protein values closer to one another, allowing for more unbiased downstream statistical testing. Secondly, normalize all protein intensities to the ratio of the median protein intensities for a given TMT channel to the median of all protein intensities. This will account for any slight mixing errors from each sample.
1. Different cell types will adhere with different strengths, so one must be careful not to dislodge the cells prematurely, depending on cell type.
2. The cell pellets can be frozen here at −80 °C for future use.
3. A 5:1 ratio of lysis buffer:cell pellet or greater is helpful here in order to make the lysate less viscous.
4. It works best to do this slowly so as not to create excess bubbles.
5. Depending on the viscosity of the lysate, there may or may not be a visible cell pellet that clearly separates from the supernatant. If there is not, move forward with the entire mixture.
6. The goal of the DTT is to reduce all disulfide bonds.
7. The goal of the IAA is to alkylate free thiols.
8. This precipitation technique requires enough protein to visualize the protein pellet. If the protein output is too low, a TCA precipitation is preferred.
9. If the pellet is difficult to disrupt, rake the tube against an Eppendorf tube rack until it is dislodged.
10. While loss in the previous precipitation steps will be unbiased, loss at this stage will be biased towards hydrophobic peptides and should be carefully avoided.
11. The protein pellets can be frozen here at −80 °C for future use.
12. Depending on the size, it may be difficult to fully resuspend the pellet. Use a small pestle to grind the pellet and disrupt as best as possible.
13. Any pellet is undigested protein. The pellet can be stored at −20 °C and further digested in the future in the case of low peptide yield.
14. SepPak columns are used for larger peptide amounts. For low amounts (<10 μg), use StageTips (packed with C18 solid-phase extraction disks [Empore]) instead to minimize peptide loss.
15. Do not let column go completely dry until indicated.
16. It may be easier to open other ports on the vacuum manifold to allow a slower draw on the column.
17. Make sure to add the 40 % ACN solution first, followed by the 80 % ACN solution, to prevent too many peptides from eluting at once and clogging the column.
18. Prepare the protein quantification assay of your choice. We use the BCA assay from Pierce.
19. More concentrated samples may be diluted 1:3–1:10 to fall into the standard curve range.
Amanda Edwards and Wilhelm Haas
20. It is very important here that the ACN used is anhydrous, as hydrated ACN will reduce the labeling efficiency.
21. The manufacturer recommends performing TMT labeling using a triethylammonium bicarbonate (TEAB) buffer. However, it has been shown previously [24] that using this buffer produces unidentified and unwanted site reaction products (in particular, singly charged ions with m/z of 303.26, 317.26, and 331.29) of high intensity in the LC-MS2/MS3 chromatograms. The formation of these products is avoided by using the described buffer conditions.
22. The given retention time range is based on the described system. This range may differ slightly between different HPLC systems and the fraction collection retention time frame should be adjusted accordingly.
23. The Orbitrap Fusion allows acquisition of high-resolution, MultiNotch MS3 data [25]. As demonstrated previously [24, 25], this approach reduces the observed interference effect in quantitation at the MS2 level. However, not all instrumentation allows for this approach. Other approaches to decrease interference in the quantitation include TMTC quantitation [31] and ion-ion chemistry for removing contaminant ions [23]. If these approaches are untenable, one must use the resulting quantitative data with appropriate caution.
References
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3. Wang Z, Gerstein M, Snyder M (2009) RNASeq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63
4. Li G, Burkhardt D, Gross C et al (2014) Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell 157:624–635
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6. Stingele S, Stoehr G, Peplowska K et al (2012) Global analysis of genome, transcriptome and proteome reveals the response to aneuploidy in human cells. Mol Syst Biol 8:608
7. Dephoure N, Hwang S, O’Sullivan C et al (2014) Quantitative proteomic analysis reveals posttranslational responses to aneuploidy in yeast. Elife 3, e03023
8. Wu Y, Williams E, Dubuis S et al (2014) Multilayered genetic and omics dissection of mitochondrial activity in a mouse reference population. Cell 158:1415–1430
9. Zhang B, Wang J, Wang X et al (2014) Proteogenomic characterization of human colon and rectal cancer. Nature 513:382–387
10. Gygi S, Rist B, Gerber S et al (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17:994–999
11. Ong S, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386
12. Syka J, Marto J, Bai D et al (2004) Novel linear quadrupole ion trap/FT mass spectrometer: performance characterization and use in the comparative analysis of histone H3 posttranslational modification. J Proteome Res 3:621–626
13. Olsen J, de Godoy L, Li G et al (2005) Parts per million mass accuracy on an Orbitrap mass
spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 12:2010–2021
14. Hsu J, Huang S, Chow N et al (2003) Stableisotope dimethyl labeling for quantitative proteomics. Anal Chem 75:6843–6852
15. Wilson-Grady J, Haas W, Gygi S (2013) Quantitative comparison of the fasted and refed mouse liver phosphoproteomes using lower pH reductive dimethylation. Methods 61: 277–286
16. Wu Y, Wang F, Liu Z et al (2014) Five-plex isotope dimethyl labeling for quantitative proteomics. Chem Commun (Camb) 50: 1708–1710
17. Blagoev B, Ong S, Kratchmarova I et al (2004) Temporal analysis of phosphotyrosinedependent signaling networks by quantitative proteomics. Nat Biotechnol 22:1139–1145
18. Thompson A, Schäfer J, Kuhn K et al (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75: 1895–1904
19. Ross P, Huang Y, Marchese J et al (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154–1169
20. Choe L, D’Ascenzo M, Relkin N et al (2007) 8 ‐ Plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer’s disease. Proteomics 7:3651–3660
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25. McAlister G, Nusinow D, Jedrychowski M et al (2014) MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal Chem 86:7150–7158
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Chapter 2
Sample Preparation Approaches for iTRAQ Labeling and Quantitative Proteomic Analyses in Systems Biology
Christos Spanos and J. Bernadette Moore
Abstract
Among a variety of global quantification strategies utilized in mass spectrometry (MS)-based proteomics, isobaric tags for relative and absolute quantitation (iTRAQ) are an attractive option for examining the relative amounts of proteins in different samples. The inherent complexity of mammalian proteomes and the diversity of protein physicochemical properties mean that complete proteome coverage is still unlikely from a single analytical method. Numerous options exist for reducing protein sample complexity and resolving digested peptides prior to MS analysis. Indeed, the reliability and efficiency of protein identification and quantitation from an iTRAQ workflow strongly depend on sample preparation upstream of MS. Here we describe our methods for: (1) total protein extraction from immortalized cells; (2) subcellular fractionation of murine tissue; (3) protein sample desalting, digestion, and iTRAQ labeling; (4) peptide separation by strong cation-exchange high-performance liquid chromatography; and (5) peptide separation by isoelectric focusing.
Key words Proteomics, Mass spectrometry, iTRAQ, Subcellular fractionation, High-performance liquid chromatography, Isoelectric focusing
1 Introduction
Quantitative analysis of protein expression, function, and subcellular localization is fundamental to network biology. Mass spectrometry (MS)-based quantitative proteomic approaches have evolved rapidly in the last 15 years and are generating datasets essential for systems biology and the modeling of biological networks [1]. Discovery applications in MS-based proteomics have largely employed untargeted strategies where proteins in one or more samples (diseased or treated) are quantified relative to the amount of proteins in a separate sample (normal or control). Quantification of the peptides/proteins can either be done by comparative analysis of spectral features in a “label-free” workflow or alternatively be accomplished through isotopic labeling or the incorporation of a differential mass tag. Isobaric tags for relative and absolute
quantitation (iTRAQ) are widely used amine-specific, stableisotope reagents which can be used to label the N terminus and ε side chain of lysines of peptides generated by tryptic digestion of extracted proteins. The reagents were designed for “multiplexing,” and four or eight separate iTRAQ labels are available permitting the simultaneous analysis of multiple samples. The labels consist of a low-mass reporter group, a balance group, and an amine-reactive group; they have isobaric masses in MS mode (145 and 305 Da for 4-plex or 8-plex reagents), but upon fragmentation release low-mass reporter ions (m/z values of 114.1, 115.1, 116.1, 117.1 for 4-plex, plus 113.1, 118.1, 119.1, 121.1 for 8-plex) allowing quantification at the MS/MS level. Unlike metabolic incorporation of stable isotopes, a key advantage to iTRAQ labels is that samples from any source, including patient material, can be chemically labeled. This fact, in combination with the ability to simultaneously analyze multiple samples, has likely contributed to the widespread use of these reagents [2, 3]. It should be noted that quantitation by iTRAQ is not perfect; contamination during precursor ion selection, specific to MS/MS quantitation, results in compression of the iTRAQ ratio and underestimation of relative protein abundance estimates; and variance is higher for low-intensity signals [4, 5]. Data processing and instrument-specific approaches aimed at addressing issues related to the precision and accuracy of iTRAQ quantitation continue to evolve and have been reviewed elsewhere [2, 3].
Technological advances in high-resolution MS instrumentation means that almost complete coverage of unicellular organisms such as yeast is now possible [6] and comprehensive analysis of mammalian proteomes is envisaged as feasible in the near future [7]. However, the required technology is not widely available and currently most researchers will find that proteome coverage is dependent on reducing sample complexity and their choice of multiple sample preparation steps including protein separation, digestion, and peptide fractionation steps. Proteomic workflows can be complex and the role of error propagation through multiple handling steps should not be underestimated [8]. Critical to the success of an iTRAQ experiment is the use of equal sample amounts, reproducible protein digestion, and efficient peptide labeling. Protein samples may be pre-fractionated either by subcellular fractionation or based on size by polyacrylamide gel electrophoresis. Proteins are most typically digested using trypsin, although other proteases can be used [ 9 ], and digested peptides are separated by either high-performance liquid chromatography (HPLC) or isoelectric focusing.
We have used iTRAQ reagents to examine differential protein expression in fatty acid-treated hepatocarcinoma cells and liver tissue mice fed a high-fat diet. Here we describe our methods for: (1) total protein extraction from immortalized cells; (2) subcellular
2 Materials
2.1 Protein Extraction from Cells
2.2 Protein Extraction from Liver Tissue
2.3 Protein
Desalting, Digestion, and iTRAQ Labeling Sample Preparation for
fractionation of murine liver tissue; (3) protein sample desalting, digestion, and iTRAQ labeling; (4) peptide separation by strong cation-exchange (SCX) HPLC; and (5) peptide separation by isoelectric focusing
All reagents should be of analytical grade and solvents either HPLC or LC-MS grade. All solutions should be prepared with distilled, deionized water (ddH2O), typically 18.2 MΩ·cm at 25 °C, except for LC-MS/MS buffers which require LC-MS-grade H2O.
1. Phosphate-buffered saline (PBS; 1×): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4.
2. Radioimmunoprecipitation assay buffer (RIPA): 150 mM NaCl, 1.0 % IGEPAL® CA-630, 0.5 % sodium deoxycholate, 0.1 % SDS, and 50 mM Tris, pH 8.0.
3. Protease inhibitor cocktail-EDTA free (PI): Mixture of several protease inhibitors inhibiting serine, cysteine, but not metalloproteases.
4. Low-speed swinging bucket centrifuge.
5. Refrigerated table top microcentrifuge.
6. QIAshredder columns (QIAGEN, UK).
7. BCA assay.
1. HEPES, EDTA, mannitol (HEM) buffer: 20 mM HEPES, 1 mM EDTA, 300 mM mannitol.
2. Protease inhibitor cocktail EDTA free (PI): Mixture of several protease inhibitors inhibiting serine, cysteine, but not metalloproteases.
3. 10 ml glass Dounce homogenizer with pestle attached to elec tric drill.
4. Refrigerated swinging bucket centrifuge.
5. Ultracentrifuge.
6. Appropriate polyallomer tubes for ultracentrifugation.
1. 2 ml ZEBA columns (Pierce Scientific, UK; see Note 6).
2. RIPA buffer and PI as before.
3. Vacuum centrifuge (Eppendorf, UK).
4. Low-bind microcentrifuge tubes (see Note 7).
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work, we would again go downstairs, and all have tea together, and after that a dance; and we would dance reels and jigs, and hornpipes, and quadrilles, but mostly reels.—Hark! Aunt Sharpshins is ringing at the door!’ And away the two little girls ran scampering upstairs; and in her haste and terror Ellen gave my head such a knock against the banisters! But she was so sorry, and kissed me so often to make it well.
Up to this time I had never been properly dressed; for, excepting a strip of bright blue ribbon round my waist, and a small cap of purple silk stuck on the back of my head, I was in the very same long white night-gown which Ellen had made for me when I first went to the pastry-cook’s house, and in which I made my first appearance in the shop among all the gas-lights and cakes on Twelfth-night. So my dear mamma and Nanny Bell determined to make me a proper dress.
CHAPTER V
MY FIRST FROCK AND TROUSERS
T were plenty of little odds and ends of silks, and stuffs, and velvets, and muslins, which Ellen had already collected, and which her aunt had told her she might have; and with these they knew they could make me a beautiful dress. They finished their dinner as fast as possible, and ran upstairs again, in order to be alone for this pleasant work.
They accordingly began by carefully measuring me round the waist and round the shoulders; and then across the back down to the waist, measuring from the right shoulder crossing down to the middle of the left side, and from the left shoulder across to the middle of the right side. Their little fingers were busy about me in all directions; they did so tickle me!
Then they measured my arms; first from the top of the shoulder to the elbow when bent, and next from the tip of the elbow to the wrist. Lastly, they measured me from the back of my neck down to the middle of the waist, just where there is, or ought to be, the most bend in a doll’s back; and from this they measured for the skirt right down below my knees, and for the trousers they measured down as low as my ankles.
But how were these two little girls to find time to do all this work for me? The whole day they were engaged, from six o’clock in the morning till bedtime. So, as it was now summer, and quite light at five o’clock, Ellen and Nanny both determined to get up at that time, and thus have an hour every morning to themselves, in order to make me a frock and trousers. And they calculated that by doing this for a week, they could easily finish the task they had set themselves.
But the poor little girls had to work so hard for more than thirteen hours every day, that neither of them could awake in time. After
several mornings, however, Ellen did manage to wake up enough to speak, and call Nanny; and Nanny woke up enough just to answer. After which down sunk their cheeks upon the pillow, and they were fast asleep in a moment.
The next morning Nanny Bell called Ellen at about half-past five o’clock, and Ellen made a great effort, and sat up in bed with her eyes shut. At last she half opened one eye, and then she saw poor Nanny as fast asleep again as if she had never called her. So back fell Ellen upon her pillow.
Now, for several nights, they both made great resolutions before they went to sleep; but when the morning came they could not keep them, though they tried very much to do so; and one morning Ellen, directly Nanny called her, rolled herself out of bed upon the floor. But there she lay, and when the other girls were going past the door to their work at six o’clock, and came in to see if Ellen and Nanny were dressed, there they found Ellen fast asleep upon the floor in her night-gown.
Something, however, happened in consequence of this. Ellen had caught a bad cold and sore throat with sleeping upon the floor, and the doctor said she must remain in bed for two days to get rid of it. Ellen was, therefore, sent to bed again soon after dinner, and as it was necessary that somebody should be with her to give her medicine or barley water to drink, Nanny Bell was chosen by her own request. Here was a chance! Now was the time to work at my frock and trousers.
But there was something to be done first. There was physic to be taken. It was brought by Aunt Sharpshins in a teacup, and it had a dark red and yellow color, and oh, such a strong smell! Poor Ellen looked at her aunt so pitifully, as much as to say, ‘Must I really take this nasty physic?’—then she looked into the teacup, and made a face—then she looked round the room making the same face, only sadder—then she gave a little frown as much as to say, ‘Why should I be afraid? I know it is good for me—I am determined to take it!’ then she shut her eyes—put the teacup to her lips—and down went the physic!
As soon as Mrs. Sharpshins left them, Nanny produced some sugarplums out of a little paper for Ellen to take after her medicine; and as they ate the sugar-plums, Nanny laughed at the horrid faces my mamma had made before she took her physic and just after it was down, and then they both laughed very much.
Ellen now sat up in bed, and Nanny helped her to prop herself up with pillows at her back, and covered her shoulders with a large shawl. Nanny then brought all the bits of muslin, and silk, and stuffs, and velvet, together with a pair of scissors, and needles and thread, and spread them out upon the quilt before Ellen. I was placed on the bed beside her with my head raised high, so that I might see them working. When all was ready, Nanny got upon the bed and sat down opposite to Ellen, and to work they both went.
The measurements had already been made, and the slips of paper with the marks were laid upon the quilt. Then they began cutting out. First they cut out my under-clothes, and these were all of cambric muslin, which they said was necessary, in order to be soft to the skin of such a little creature as I was. I could not help laughing to myself when I heard them say this, because I was made all of wood, and my skin was only the fine, hard, polished varnish of the celebrated Mr. Sprat. I was not quite so tender as they fancied. They next cut me out a small under-bodice of white jean instead of stays. Then came the trousers, which were cut long and full, and were of soft white muslin trimmed with open work. They then cut out a petticoat of fine cambric muslin, the body quite tight and the skirt very full all round. My frock was made to fit nicely to the shape, but not too tight. It was of fine lemon-colored merino, with a sash of violet-colored velvet, and very full in the skirt, and they said it must have some stiff muslin inside the hem to make it set out, and not hang too loosely in the folds.
When all was cut out and arranged, my mamma and Nanny both went to work with their needles, and they worked all the day as long as they could see. The under-clothes and the trousers were all quite finished, and the body and one sleeve of the frock was begun.
The next morning, after my mamma had taken her medicine and made the same horrid face as before, only not quite so bad this time, they went to work again. But this second morning the weather was not so warm as the day before; so Nanny went to the bed of one of the other girls and took off the top sheet, and tied up a bit of it in the middle with a long and strong tape in a strong knot, and then with a chair upon the bed she managed to tie the other end to a nail in the wall just over the head of the bed; she then spread out all the sheet that hung down so as to cover them both in, like a little tent. And in this pleasant manner they worked all the second day, by which time my frock was quite finished.
They Worked as Long as They Could See.
Besides this they had made me a pair of silk stockings, which were sewed upon my legs to make them fit better; and as I was naturally from my birth rather stiff in the ankles and instep, they made the stockings without feet, but sewed black satin over both my feet in the shape of the prettiest boots possible, with stitches of cross-work in front. When all was done, and everything put upon me, nothing would do but they must take me out for a walk round the room.
Out we all got from the tent; my mamma in her night-gown and shawl, with a bit of flannel round her throat, and list shoes, and I walking between the two little girls, each holding me by the hand. But we had hardly walked twice round the room, talking like ladies who are out in the park, when suddenly we heard Aunt Sharpshins coming upstairs! In a moment we were all upon the bed—down came the tent—underneath the bed it was thrown—into the bed we all three got as quickly as possible—and when Mrs. Sharpshins came into the room we all seemed fast asleep!
She stood at the foot of the bed, looking at us. After a minute or two she went down again.
‘How you laughed and shook the bed,’ said my mamma to Nanny. ‘I thought she would have found us out, and somehow I wished she had. I don’t like to have pretended to be asleep.’
‘But,’ answered Nanny, ‘she would have been so unkind if she had seen us walking in the park.’
‘I wish people would not be unkind,’ sighed my mamma; and then she added, ‘How dear and kind you are, Nanny; and how you have worked for me, and nursed me all these two days.’
At this they threw their arms round each other’s necks, and so we all three went to sleep in reality, quite forgetting the tent which had been thrown under the bed. But it was a good-natured, merry girl that it belonged to, and she only gave my mamma and Nanny a good tickling when she found it, after a long search, at bedtime.
CHAPTER VI
THE
LITTLE LADY
M mamma got quite well as soon as my frock and trousers were finished; and whenever she was allowed to go out with her aunt she took me with her. The girl whose sheet had been taken for the tent had made me a scarf of violet-colored satin, and a white silk bonnet, and these I always had on when we went out.
In a few weeks, however, I was destined to lose this kind mamma, and become the dear doll of another. If I could have foreseen that this would happen I should have fretted very much, because I was so fond of Ellen Plummy.
But it did happen, and in this manner
One fine summer’s day Mrs. Sharpshins took Ellen for a walk in St. James’ Park, and after a little time we came to the piece of water, and saw several pretty children feeding the swans that live in that water. The children had some bread and small buns, which they broke in little pieces and threw into the water, where they floated till the swans swam up to the bread and bent their long white necks down to eat. Ellen begged her aunt to let her stay and look at the swans. ‘Look, Maria!’ said she to me, ‘what beautiful, bright, black eyes they have, and what lovely, snow-white necks, and how gracefully the breast of the swan moves upon the water, while the necks are in the shape of a beautiful arch!’
And These I Always Had On When We Went Out.
While they were looking at the swans, a very tall footman, in a green and gold livery, with a long, golden-headed cane and powdered hair came up to Mrs. Sharpshins from a carriage that was waiting near at hand, in which sat a grown-up lady with a little lady by her side. Both of them had also been observing the swans; but in doing so the little lady had at the same time fixed her eyes on me.
‘The Countess of Flowerdale wishes to speak with you,’ said the footman to Mrs. Sharpshins. Now the countess was a great lady, who sometimes employed Aunt Sharpshins to make her dresses for the country and to walk in the garden. We went with the footman, and I could see that Mrs. Sharpshins was in a great agitation.
‘Mrs. Sharpshins,’ said the countess, smiling, and with a gentle voice, ‘this young lady has been looking at your little girl’s doll more than at the swans, and she has taken a great fancy to the doll. The little girl is your niece, I believe. Will she part with her doll? I shall be glad to purchase it or send her another.’
‘Oh, anything your ladyship wishes, of course,’ said Mrs. Sharpshins, with a very low curtsey.
‘Would you like to part with your doll, my dear?’ said the lady to Ellen.
I felt Ellen give me such a close hug as much as to say, ‘Oh, no, no!’ But her aunt stooped down and looked in her face under her bonnet with such a look! The great lady did not see it, but I saw it.
‘I could not think of taking it from your little niece if she is indisposed to part with it,’ said the great lady in a sweet voice.
Upon this the small lady by her side, who seemed to be about eight years of age, turned red in the face—the corners of her mouth drooped down—her eyes grew large and round, and out rolled one large, proud tear. But she did not cry or say a word.
Whether it was this one silent tear of the little lady, or the sweet voice of the great lady, or the look that her aunt had given her under her
bonnet, I do not know, but Ellen, first giving me a kiss, lifted me up towards the carriage window, and gave me into the hands of the little lady with such a sigh!
‘Thank you, my dear,’ said the great lady, ‘I will take care to send you another handsome doll and doll’s cradle to-morrow morning, and something besides; and Mrs. Sharpshins, you can make me three or four more morning dresses the same as the last. I am in no hurry for them.’
The very tall footman got up to his place behind the carriage—the carriage drove off; the great lady nodded to Ellen; the little lady kissed her white glove to her; and Mrs. Sharpshins made a low curtsey, taking care to step just before Ellen in order that they should not see the tears that were just beginning to gush out of her eyes.
My new mamma, the little Lady Flora, was very pretty. She had a complexion like the most delicate wax-work, large bright eyes, a dimple in each cheek, and dimples all over her little knuckles. She had taken off her gloves to arrange my hair better, and began at once to talk to me in a very delightful manner.
We drove from St. James’ Park into Hyde Park, and on the way we passed a very great doll indeed, but looking so cross and black, and without any clothes on. ‘Look there, dear!’ said my little lady mamma, ‘that is the strongest and largest doll ever seen in London. His name is “Achilles,”—and the ladies of London had him made of iron and brass, because the Duke of Wellington was so lucky in playing at ball on the fields of Waterloo!’ The countess seemed much amused with this account. We met a great number of elegant carriages on our way, and nearly all the ladies inside exchanged salutations with the countess, and nodded to my little lady mamma. All who were elegant, and richly dressed, and beautiful, and in fine carriages with rich liveries, seemed to know each other, and to be upon such delightful terms of affectionate intimacy! ‘Oh!’ thought I, ‘here is a new world! Everybody seems to respect, and admire, and love everybody else! How very delightful!’
CHAPTER VII
THE WEST END OF THE TOWN
O house was in Hanover Square, a few doors from the Queen’s Concert Rooms. There happened to be a morning concert on the first day of my arrival, and as one of the drawing-room windows, where I sat with my little lady mamma, opened out upon the balcony, we could every now and then hear the trumpets and drums, and one violin which squeaked so sweetly high above all the rest.
At four o’clock my new mamma went out for a drive in her carriage with her governess, and chiefly to buy several things for me. Of course, I went too.
First we drove to pay a visit to a young lady in Grosvenor Square, and after this we drove to a toy-shop in Oxford Street, and there the little Lady Flora bought me a cradle of delicate white basket-work, with a mattress and pillow covered with cotton of pale pink and lilac stripes. She wanted a feather-bed; but they had not got one. The governess then bought a large, handsome doll, chosen by Lady Flora, to send to my dear first mamma, Ellen Plummy, in exchange for me, and also a nice cradle, and one or two other things which I did not see.
We next went down Regent Street, and sent the very tall footman with the gold-headed cane and powdered hair into every shop that seemed likely, to ask if they had a doll’s feather-bed. But none of them had. One young person, however, dressed in black, with a pale face, and her hair very nicely plaited, came out to the carriage window and said, ‘They would be most happy to make a feather-bed for the doll, if her ladyship would allow them that honor!’ My little lady mamma, however, said, ‘Certainly not—I thank you.’
We passed the Regent’s quadrant, after sending into two or three shops, and then turned up Piccadilly, and got out at the Burlington
Arcade. But no such thing as a doll’s feather-bed could be found. The little lady, however, bought me a small gold watch and chain, which cost a shilling. We then returned to the carriage, drove down Waterloo Place, and sent into several shops to inquire, while we slowly drove towards the Duke of York’s column. My lady mamma explained to me that the black doll on the top was once a great duke, who was at the head of all the army when he was alive, in the same way that he was now at the top of that fine column. The very tall footman presently returned, saying he was very sorry to inform Lady Flora that he had not been so fortunate as to discover a doll’s feather-bed at any of the shops; so we turned round and drove up Bond Street, and tried at several shops with no better success; then we passed again down Oxford Street, and went to the Soho Bazaar. There, at the top of a long room—on the left-hand side—in a corner —there, at last, we did find a doll’s feather-bed, and of a very superior quality. No doll in the world, and particularly a wooden doll, could have wished for anything softer. At the same place were also many articles of furniture, such as dolls of the higher class are accustomed to have, and some of these were bought for me. That which I was most pleased with was a doll’s wardrobe made of cedar wood, with drawers for clothes in the middle, and pegs to hang dresses upon at each side, and all enclosed with folding doors, and smelling so sweet. All of these things being carefully packed up in silver paper, and then placed one upon the other, were given to the very tall footman with powdered hair, who, receiving them with a serious face, and carrying them balanced on the palm of one hand, and holding up his long gold-headed cane in the other, slowly walked behind us, with his chin raised high out of his white neckcloth, to the admiration of everybody in the bazaar, as we returned to our carriage.
We now drove once more into Regent Street, to a pastry-cook’s, and there I was left lying upon the seat of the carriage all alone, while Lady Flora and her governess went to have something nice. But I did not care much about this, as my mind was occupied with several thoughts. In the first place, the pastry-cook’s window, though very elegant, presented nothing like the brilliant appearance of Mr.
Plummy’s shop-window on Twelfth-night! No—that first impression exceeded anything else of the kind, and was never to be effaced. But there was one other thought that troubled me a little. It was this. I had been accustomed hitherto to think myself not only very pretty, but one of the very nicest and best dolls that could possibly be. I had always understood that the celebrated Mr. Sprat, who had made me, was one of the very first doll-makers in England! The master of the doll-shop in Holborn, who had walked to and fro, like Napoleon Bonaparte in a brown paper cocked hat, had said so in my hearing, and I had believed it. I naturally considered myself a charming doll. But I had seen many other dolls of quite a different make in the Soho Bazaar!—dolls which I could not help fancying were superior to any of those made by poor Mr. Sprat, and therefore very superior to myself. This thought hurt my vanity and humbled me. Of course I had been very vain and conceited. What else could you expect of a doll? But now I certainly felt much less vain, for I plainly saw that there were other dolls in the world who were far prettier and better made than myself. However, as I had been already beloved by two mammas, I soon became contented, and felt no jealousy or envy of the prettiness or fineness of others; and I also believed that if I was amiable, and could become clever, I should never be without somebody to love me.
My mamma and her governess now returned to the carriage and we drove home.
CHAPTER VIII A NARROW ESCAPE
I a narrow escape from a most terrible accident a few days after, of a kind which I shall never forget as long as I live. As it happened at the close of a day on which I saw several new things, I may as well give a short account of that day, and finish with my narrow escape.
The carriage was ordered at twelve o’clock, and we drove to Regent’s Park. I had on a new bonnet with a white lace veil, and looked very nice. After driving once round the circle, we got out at the Zoological Gardens, and went in to see the animals.
My little lady mamma first took me to see the parrots, and parroquets, and macaws. Some of the macaws were all white; some white, with an orange-colored topknot; some were green, with scarlet and blue in the wings and tail, and with scarlet and white in their faces. Then they had two or three very long, straight feathers in their tails and they spoke to each other, and often scolded in a very hoarse voice. Some of the parrots were all green, some all grey; but there was one of the parroquets—a little bright-eyed, quick fellow,— who was nearly all red, and had a funny, impudent crown of feathers of white and purple upon the top of his head, but a very short tail. Now, as we were looking at him, Lady Flora suddenly took a fancy to touch his short tail—not with her own hand though, but with mine, which she poked through the wires of his cage for that purpose. ‘Kark!’ cried the little red, quick fellow, turning round very briskly and giving such a peck at my hand. He just missed me, because the governess, who was close by, instantly drew back my mamma’s arm and mine too, of course, at the same time; the peck, however, fell upon the edge of the cage and made a mark in the wood. This was a narrow escape, everybody would say; still it is not the terrible one I shall presently have to relate.
After this, the same little quick fellow pretended his poll wanted scratching, and held down his head to have it done for him, with his eyes shut—one eye, though, not quite closed—and his head turned rather sideways. ‘No, no!’ said the governess, ‘no, thank you, sir; you only want to get another chance of a peck at our fingers!’ So we went away, and then the little quick fellow looked up in a moment with such a bright eye, and cried, ‘Kark! skrark!’
After this my mamma took me, all trembling as I was, to see the monkeys. As she remembered the danger I had been in from the red parroquet with the impudent topknot, Lady Flora did not put either of my hands into any of the cages, but held me up in front of one of them, that I might see the monkeys. Oh, how I wished for a voice to cry, ‘Not so close, mamma! Do not hold me so close!’
The monkey who was nearest to the bars was the quietest of them. While the others were running and skipping, and climbing all over the cage, this one sat quite still, with his head bent down and his eyes looking upon the floor; and now and then he looked into the black palm of his little brown hand, with a very grave and earnest face, as if he was considering something about which he was very anxious: when all of a sudden he darted one arm through the bars of his cage, right at my head, and just reached my white veil with his little brown hand! He tore it quite off from the bonnet—ran up the wires in front, squeaking and chattering—and the next moment we saw him at the back of the cage, high up, sitting upon a small shelf tearing my veil all to pieces, and showing us his white teeth, with round staring eyes, and his mouth opening and shutting as fast as possible.
This also was a narrow escape, everybody will say; still it is not the terrible one I shall almost directly have to relate.
We went to see the tigers and leopards, and while the governess was looking at a zebra, we went too close to be safe, and also too close to the bars of the elephant’s enclosure, so that he could have reached us very well with his trunk; but none of these chances are like the terrible escape I am now about to relate. I may well call it a terrible one, because I might have broken my neck or my back, or
